Topical administration of amifostine and related compounds

ABSTRACT

The present invention is directed to methods of treating or protecting mucosal tissue from damage associated with radiation and/or chemotherapeutic treatment of cancers, by the topical application of amifostine and related compounds. These methods avoid the side effects of systemically applied radio/chemo protectants. The invention is also directed to treatment and prevention of infections associated with mucositis by topical application of amifostine and related compounds.

[0001] This application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/083,071, filed Apr. 27, 1998.

1. FIELD OF THE INVENTION

[0002] The invention relates to topical use of aminothiols andcompositions containing them for the protection from and treatment ofradiation and/or chemotherapeutic damage to tissue.

2. INTRODUCTION

[0003] The present invention is directed to novel methods of usingamifostine and structurally related aminothiols, includingaminophosphorothioates, and their metabolites for the topical treatmentof tissue. In particular, the invention relates to protecting mucosal,skin or hair tissue from damage by radiation and chemotherapeuticagents, as well as to the treatment of such damaged tissue. In oneaspect, the invention encompasses methods of protection of mucosaltissue, and especially mucosal tissue of the head and neck regions, fromchemical, radiation, and radio/chemo induced mucositis and conditionsrelated to mucositis, associated with the treatment of cancers. Themethods are achieved by the topical application of amifostine,structurally related compounds or their metabolites. The invention alsoencompasses treatment and prevention of infections associated withmucositis in mucosa of the head and neck region by topical applicationof amifostine and related compounds. Topical application of theseradiochemical protectants allows the use of more aggressiveradiochemical treatment schedules while avoiding the toxic effects ofsystemically administered amifostine.

3. BACKGROUND OF THE INVENTION

[0004] 3.1 Systemically Administered Amifostine

[0005] The compound S-2-(3-aminopropylamino)ethyl dihydrogenphosphorothioate (also known as amifostine, ethiofos, Ethyol®, NSC296961, and WR-2721, and which will hereinafter be referred to as“amifostine” has been known for over thirty years, and was originallydeveloped by the Walter Reed Institute of Research as an antiradiationagent (radioprotectant) In particular, amifostine was developed formilitary use against x-ray or nuclear radiation which may be encounteredduring military conflicts. Bulk amifostine and other aminoalkyldihydrogen phosphorothioates, and methods to obtain them, are disclosedin U.S. Pat. No. 3,892,824, which is incorporated herein by reference.

[0006] In addition to its utility as a military antiradiation agent,amifostine has demonstrated excellent utility as a non-militaryradioprotectant and chemoprotectant when administered systemically priorto chemotherapy and/or radiation therapy. Amifostine acts to protectnormal tissue against the adverse effects which accompany the use ofradiochemical therapies for the treatment of various cancers, whilelargely leaving the target cancerous tissues unprotected. Thisprotective effect is observed in radiation and chemotherapeutictreatments by, for example, alkylating agents such as cyclophosphamide,cisplatin, carboplatin, doxorubicin and its derivatives, and mitomycinand its derivatives. For representative studies, see, e.g., Constine etal., Int. J. Radia. Oncol. Biol. Phys., 12, 1505-1508 (1986); Liu etal., Cancer, 69(11), 2820-2825 (1992); Wadler et al., J. Clin. Oncol.,11(8), 1511-1516 (1993); and Buntzel et al., Ann. Oncol. 7 (Suppl. 5),81, 381P (1996).

[0007] Similarly, it has been reported that amifostine may be used toprotect against the harmful side effects of 3′-azido-3′-deoxythymidine(AZT) therapy. In addition, amifostine and its derivatives appear toexert their protective effects without significantly affecting thebeneficial properties of the administered therapeutic agents.

[0008] Amifostine is approved in the United States for treatment toreduce the cumulative renal toxicity associated with repeatedadministration of cisplatin in patients with advanced ovarian ornon-small cell lung cancer. Physicians' Desk Reference, 51st ed. (1997).

[0009] Amifostine is a pro-drug that is dephosphorylated at the tissuesite by alkaline phosphatase to the free thiol, which is the activemetabolite (WR-1065). Without wishing to be bound by theory, it isbelieved that once inside the cell, the active free thiol can protectagainst the toxicities associated with radiation by acting as ascavenger for oxygen free-radicals. (See, Yuhas, in Radiation-DrugInteractions in Cancer Management, pp. 303-352 (1977); Yuhas, J. Natl.Cancer Inst., 50, 69-78 (1973); Philips et al., Cancer Treat. Rep., 68,291-302 (1984)).

[0010] Amifostine shows these favorable radioprotective effects whenadministered systemically prior to radiation treatment. Systemicadministration, however, suffers from numerous disadvantages. Thetypical systemic route of administration is intravenously, butadministering compounds intravenously is extremely inconvenient,particularly when a daily dosing schedule for several weeks isnecessary. In addition, when amifostine is administered systemically,patients suffer from dose-dependent undesirable side-effects such asnausea, vomiting, emesis and hypotension, as well as flushing or feelingof warmth, chills or feeling of coldness, dizziness, somnolence, hiccupsand sneezing. At high enough doses, systemic amifostine is toxic.

3.2 Topically Administered Amifostine

[0011] Topical administration of amifostine, if feasible, would beadvantageous for a number of reasons. The therapeutic effect ofradiation is dose-dependent, so that it would be desirable in many casesto increase the radiation dosage, or use an accelerated radiationschedule, in order to increase the cure rate. Such increased doses ofradiation, however, require corresponding increases in amifostine dosesin order to counteract the damage to normal tissue accompanying theincreased or accelerated radiation schedule. The protective effect ofthe compound is said to be dependent upon the concentration ofamifostine or its active metabolite present in the normal tissue.Because of the adverse side effects of systemic amifostine, however, theamount that can be administered systemically is severely limited.Topical administration would allow greater local control of theamifostine concentration, allowing higher local concentrations withoutdelivery of the higher doses to unaffected tissues and organs. To date,however, topical administration of amifostine has not been shown to befeasible.

[0012] The need for an effective topical radiation and chemotherapy(“radio/chemo”) protectant is especially acute in patients sufferingfrom radiation or chemically induced damage to mucosal tissue, such asmucositis and conditions associated with mucositis. As a specificexample, cancers of the head and neck are often highly localized, andwould benefit from aggressive radio/chemo treatment. The normal mucosaltissues of the head and neck region, such as the oral mucosa, aresusceptible to chemical and radiation damage. Chemical, radiation, andcombined radiation and chemical treatment act to deplete the mucosalbasal epithelium, thinning the tissue and causing inflammation,swelling, erythema and ultimately ulceration.

[0013] Ulceration of the mucosa leads to additional complications, asthe exposed underlying tissue is vulnerable to infection. For example,Bourhis et al. evaluated an accelerated radiation schedule in patientssuffering from locally advanced head and neck cancers. Bourhis et al.,Int. J Radiat. Oncol. Biol. Phys., 32(3), 747-752 (1995). In all of thepatients treated with the accelerated schedule, confluent mucositis wasobserved, and more than half of the patients required hospitalization totreat the mucositis. Similar results were reported by Delaney et al.(96% showed confluent mucositis), following a different aggressiveradiotherapy schedule. Delaney et al., Int. J Radiat. Oncol. Biol.Phys., 32(3), 763-768 (1995). But for the sensitivity to head and neckmucosal tissue to radio/chemo damage, more aggressive therapeutictreatments including increased radiation doses and accelerated radiationschedules could be particularly effective at treating cancers in theseregions. Thus, protection of mucosal tissue of the head and neck regionswould be especially advantageous.

3.2.1 Topical Application to Non-Mucosal Tissue

[0014] Although much is known about the radioprotective effects ofsystemically administered amifostine and related compounds, relativelylittle is known about the effects of these compounds when administeredtopically. The few studies which have addressed topical administrationhave produced inconclusive results.

[0015] In an early study, Utley et al. found that topical administrationof amifostine in carbowax to the oral mucosa of mice subjected to wholehead irradiation prevented oral radiation death syndrome (LD50/8-10) bya factor of 1.4, with no toxicity observed at the dosages tested. Utleyet al., Int. J. Radiat. Oncol. Biol. Phys., 1, Supp. 1, No. 154 (1976).Systemically administered amifostine was found to be more effective inpreventing oral radiation death, although some deaths due to drugtoxicity were reported. The study did not address protection of othertissues or of the oral mucosa per se from mucositis.

[0016] Several studies have looked at the radioprotective effects oftopical amifostine on radiation-induced damage to non-mucosal tissue,particularly to rat and mouse skin.

[0017] In an early pre-clinical study, Lowy et al. studied theradioprotective effects of systemically and topically administeredamifostine in mice. Lowy et al., Radiation Biology, 105, 425-428 (1972).The amifostine was administered to mouse skin as a paste formed from anaqueous sodium bicarbonate solution (pH 7) and Unibase. Although thestudy found systemically administered amifostine to be effective inreducing the severity of radiation damage, topically applied amifostinewas found to provide no radioprotective effect at any dose studied.

[0018] Similarly, Verhey et al. found amifostine to be ineffective toprotect mouse skin from radiation-induced damage when applied topically.Verhey et al., Radiation Research, 93, 175-183 (1983). A gauze saturatedwith a 10% solution of amifostine in saline was applied to murine skinfor 15 to 60 minutes, followed by ¹³⁷CS irradiation. The study found nosignificant radioprotective effects for topically-applied amifostine.

[0019] More recently, Geng et al. compared the effects of topically andsystemically administered 16,16 dm prostaglandin E₂ (PGE₂) andamifostine on radiation-induced alopecia in mice. Geng et al., Int. J.Radiat. Biol., 61(4), 533-537 (1992); see also Malkinson et al., J.Invest. Dermatol., 101(suppl), 135S-137S (1993), reporting similarresults. In the topical studies, a 0.3 mg sample of the dephosphorylatedform of amifostine, WR-1065, in 0.2 mL Ringer's solution wasadministered to the depilated mouse skin prior to fractionatedirradiation treatment, then the rate of hair regrowth was studied as afunction of radiation dosage. Although topically administered WR-1065showed some effectiveness in protecting hair matrix cells fromradiation-induced injury, the Geng study found systemically administeredamifostine to be more effective at all radiation doses studied.

3.2.2 Topical Application to Mucosal Tissue

[0020] Several studies have examined the possibility of using amifostineor related compounds topically to protect intestinal mucosal tissue fromradiation damage.

[0021] Ben-Josef et al. demonstrated that amifostine applied topicallyby an intrarectal injection of a 2% amifostine gel results in anaccumulation of the amifostine metabolite WR-1065 in the rectal wall ofrats. Ben-Josef et al., Radiation Research, 143, 107-110 (1995). Thestudy did not, however, examine the radioprotective effect in mucosaltissue that might result from the accumulated WR-1065.

[0022] Montana et al. tested the effect of amifostine applied topicallyto protect intestinal mucosa in patients undergoing radiation treatmentof the pelvis. Cancer, 69(11), 2826-2830 (1992). An amifostine inProctofoam preparation was administered topically by enema at dosages of100 to 450 mg per enema 45 minutes prior to pelvic radiation treatment.The Montana et al. study found that topically applied amifostine avoidedmany of the side effects typically observed when the drug isadministered systemically. However, no significant differences werefound in rectal mucosal damage between a control group, which did notreceive amifostine, and a test group, which received the topicalamifostine treatments. Montana et al. speculated that the lack ofprotective effect may be due to the mode of administering the drug.

[0023] An opposite conclusion was reached in an animal study by Delaneyet al. Delaney et al., Cancer, 74(8), 2379-2384 (1994). Delaney et al.studied the radioprotective effect of topical solutions of amifostineinjected into the small intestine of rats. Amifostine was prepared in apH 9 Tris buffer (tris(hydroxymethyl)aminomethane) at a concentration of150 mg/mL, and administered intralumenally prior to irradiation of theexteriorized rat small bowel. The study concluded that amifostine, andparticularly amifostine in an alkaline vehicle, was an effectiveradioprotector against intestinal mucositis in rats.

[0024] Clearly, the results of these limited studies have beeninconclusive and can be considered contradictory. There is some evidencethat topical amifostine or WR-1065 may provide protection of skin orintestinal mucosa, but the studies to date show conflicting results. Inaddition, the studies of mucosal tissues to date have focused onintestinal mucosa and the effects of radiation induced mucosal tissuedamage that accompanies pelvic irradiation. Some studies suggest thatthe vehicle used to deliver the active agent may be a factor to considerin determining potential efficacy.

[0025] Mucosal tissues of the head and neck region are particularlysensitive to radiation and chemically-induced damage association withradiochemical treatment of head and neck cancers. Mucositis of thesetissues results in extreme patient discomfort, as well as incomplications due to infection of ulcerated mucositic tissues. There hasyet to be identified a safe and effective method of protecting themucosal tissues of the head and neck region from radiochemically-induceddamage without the undesirable side effects of systemic administrationof protectant drugs.

[0026] Thus, there is a need for a safe and effective method ofprotecting tissues from damage due to radio- or chemotherapeutictreatments while avoiding the undesirable effects of systemicallyadministered radio/chemo protectants.

4. SUMMARY OF THE INVENTION

[0027] The present invention relates to methods of protecting ortreating tissue, skin or hair, particularly mucosal tissue, from damageassociated with radiation treatment and chemotherapy, while avoiding theundesirable side effects associated with conventional systemicadministration of radio/chemo protectant compounds.

[0028] One aspect of the invention relates to protection of tissue bytopically administering to the tissue prior to, during, or after, orpreferably both prior to and during irradiation and/or chemotherapy, atherapeutically effective amount of an aminophosphorothioate oraminoalkyl thiol compound. The aminophosphorothioate or aminoalkyl thiolcompound has the formula:

R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂

[0029] wherein:

[0030] R₁ is hydrogen, C₁-C₇ aryl, C₁-C₇ acyl, or C₁-C₇ alkyl;

[0031] R₂ is hydrogen, PO₃H₂ or R₃, wherein R₃ isR₁NH(CH₂)_(n)NH(CH₂)_(m)S—; and

[0032] n and m are each independently an integer from 1 to 10,preferably from 2 to 6.

[0033] The invention also encompasses pharmaceutically acceptable salts,solvates and hydrates of the aminophosphorothioate or aminoalkyl thiolcompounds.

[0034] In another aspect, the invention relates to protection of mucosaltissue by topically administering to the mucosal tissue prior to,during, or after, or preferably both prior to and during irradiationand/or chemotherapy, a therapeutically effective amount of theaminophosphorothioate or aminoalkyl thiol compounds described herein.

[0035] It is surprisingly discovered that the aminophosphorothioate oraminoalkyl thiol compounds also exhibit significant antibacterialproperties. Thus, another aspect of the invention relates to a method ofprotecting tissue from bacterial infection, or treating the tissue forinfection, by topically administering to the affected tissue theaminophosphorothioate or aminoalkyl thiol compounds described herein.This is particularly advantageous given the secondary infections thatcan occur in cancer patients, particularly mucosal tissue infections inpatients undergoing treatment with radiation and/or chemotherapy.

[0036] In a further aspect, the present invention encompasses a methodof protection or treatment of tissues from radiation or chemicallyinduced damage and from infection by topically administering to thetissue the aminophosphorothioate or aminoalkyl thiol compounds describedherein. Such tissue includes hair, skin and mucosal tissue.

[0037] In another aspect, the invention relates to self-preservingmulti-dose pharmaceutical preparations which contain anaminophosphorothioate or aminoalkyl thiol compound.

5. BRIEF DESCRIPTION OF THE FIGS.

[0038] FIG. 1 shows the weight loss of mice irradiated on the lip with asingle dose of 16.5 Gy, in groups which received systemic amifostine,topical amifostine, and a control group which received no amifostine

[0039] FIG. 2 shows the lip mucosal reaction scores for the irradiatedmice.

6. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention provides for methods of treatment oftissue, hair and skin, particularly mucosal tissues, to protect thesetissues from radiation and chemically-induced damage without thewell-known side effects of systemically administered mucoprotectantcompounds. The invention arises, in part, from the surprising discoverythat the radioprotectant compound, amifostine, and structurally relatedcompounds and metabolites, can be effectively and safely appliedtopically to tissues, including mucosal tissues, to provide significantradioprotection. The specific compounds which can be used, their methodsof use, and examples of their use are described in the followingsections. These include prodrugs, metabolites, derivatives and analoguesof amifostine as well as salts, solvates and hydrates of thesecompounds.

[0041] The invention also encompasses an improved therapy for thetreatment of neoplastic disorders in humans by utilizing the topicalapplication of the compounds described herein before, during and/orafter treatment with radiation or chemotherapeutics. This topicalapplication can both treat and protect the patient from damage includingmucositis and related disorders as well as bacterial infection.

6.1. Protection By Topically Applied Amifostine And Related Compounds

[0042] In one embodiment, the present invention relates to topicaladministration of one or more aminoalkyl phosphorothioate or aminoalkylthiol compounds to tissue, skin or hair, prior to, during and/or afterirradiation and/or administration of chemotherapeutic treatments.

6.1.1 Compounds

[0043] Compounds which can be advantageously administered according tothe methods described herein are aminoalkyl phosphorothioate oraminoalkyl thiol compounds which exhibit selective radioprotection orchemoprotection of normal tissues. Such aminoalkyl phosphorothioate oraminoalkyl thiol compounds, as well as pharmaceutically acceptable saltsand/or hydrates thereof, are either known to those of skill in the artor can be identified without undue experimentation using establishedtests routinely employed in the art.

[0044] Compounds that can be used within the present invention includeamifostine (WR-2721), as well as salts, hydrates, active metabolites,pro-drugs, and functional derivatives or analogues. More specifically,the invention includes all pro-drugs and metabolites of amifostine andpro-drugs of the active metabolites. Thus, compounds known to theskilled artisan to be suitable for administration to humans and known tobe metabolites or otherwise converted into active thiols includingmetabolites such as WR-1065 and WR-33278 (disulfide), and WR-151327 andits active thiols, including metabolites such as WR-151326 and itscorresponding disulfide, are encompassed within the present invention.

[0045] Similarly, included herein are aminothiols that exhibit activitysimilar to that of amifostine or its metabolites. Preferably, thesecompounds are structurally related to amifostine. Alternatively, thesecompounds are prodrugs that are metabolized in vivo to a biologicallyactive agent. These compounds are also encompassed by the presentinvention. Specific examples are illustrated herein.

[0046] Aminothiol compounds which can be used in the present inventionare represented by the following formula (I):

(I) R₁NH(CH₂)_(n)(CH₂)_(m)SR₂

[0047] wherein R₁ is hydrogen, C₁-C₇ aryl, C₁-C₇ acyl, or C₁-C₇ alkyl;R₂ is hydrogen, PO₃H₂ or R₃, wherein R₃ is R₁NH(CH₂)_(n)(CH₂)_(m)S—; andn and m are each an integer from 1 to 10, preferably from 2 to 6.

[0048] The methods of the present invention also encompasses the use ofpharmaceutically acceptable salts and hydrates of the compounds offormula (II) above.

[0049] Preferred compounds useful in the methods of the invention arethe S-ω(ω-amino-alkylamino)alkyl dihydrogen phosphorothioate analoguesrepresented by the formula:

R—NH—(C_(n)H_(2n))—NH—(C_(m)H_(2m))—S—PO₃H₂

[0050] wherein R is hydrogen or an alkyl group containing 1 to 7 carbonatoms and m and n independently have a value of from 1 to 10, preferably2 to 6.

[0051] Amifostine (WR-2721) has the structure:

[0052] H₂N—(CH₂)₃—NH—(CH₂)₂—S—PO₃H₂

[0053] One preferred metabolite of amifostine is a dephosphorylated freethiol form known as WR-1065 (chemical nomenclature:S-2-(3-aminopropylamino) ethanethiol), which can be depicted as follows:

H₂N—(CH₂)₃—NH(CH₂)₂—SH

[0054] Another preferred metabolite of amifostine is its disulfide,known as WR-33278([2-[(aminopropyl)amino]ethanthiol]-N,N′-dithioidi-2,1-ethanediyl)bis-1,3-propanediamine),which can be depicted as follows:

H₂N—(CH₂)₃—NH—(CH₂)₂—S—S—(CH₂)₂—NH—(CH₂)₃—NH₂

[0055] A preferred analogue of amifostine is the compound designated asWR- 151327 (chemical nomenclature:1-propanethiol-3-[[3-(methylamino)propyl]amino]-dihydrogenphosphothiorate), which can be depicted as follows:

CH₃NH(CH₂)₃NH(CH₂)₃SPO₃H₂.

[0056] Another preferred analogue of amifostine is the compounddesignated WR- 151326, a dephosphorylated free thiol form of WR-151327having the chemical structure: CH₃NH(CH₂)₃NH(CH₂)₃SH.

[0057] Other specific compounds suitable for use in the presentinvention include, but are not limited to:

[0058] S-1-(aminoethyl)phosphorothioic acid (WR-638);

[0059] S-[2-(3-methylaminopropyl)aminoethyl]phosphorothioate acid(WR-3689);

[0060] S-2-(4-aminobutylamino)ethyl phosphorothioic acid (WR-2822);

[0061] 3-[(2-mercaptoethyl)amino]propionamide p-toluene-sulfonate(WR-2529);

[0062] S-1-(2-hydroxy-3-amino)propyl phosphorothioic acid (WR-77913);

[0063] 2-[3-(methylamino)propylamino]ethanethiol (WR-255591);

[0064] S-2-(5-aminopentylamino)ethyl phosphorothioic acid (WR-2823);

[0065] 1-[3-(3-aminopropyl)thiazolidin-2-Yl]-D-gluco-1,2,3,4,5pentane-pentol dihydrochloride (WR-255709).

[0066] Additional aminothiols suitable for use in the present inventioninclude, but are not limited to, S-2-(3-ethylaminopropylamino)ethyldihydrogen phosphorothioate, S-2-(3-aminopropylamino)-2-methylpropyldihydrogen phosphorothioate, S-2-(2-aminoethylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(4-aminobutylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(5-aminopentylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(6-aminohexylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(2-methylaminoethylamino)-2-ethyl dihydrogenphosphorothioate, S-2-(3-methylaminopropylamino)-2-ethyl dihydrogenphosphorothioate, and S-3-(3-methylamino-propylamino)-3-propyldihydrogen phosphorothioate (WR-151327) and pharmaceutically acceptablesalts thereof. Preferably, the aminothiol is amifostine, WR-1065,WR-33278, WR-151327 or WR-151326; most preferably it is amifostine.

[0067] Amifostine, and many of its salts, analogues and derivativesthereof suitable for use in the methods of the invention arecommercially available, or can easily be prepared using standardtechniques. The aminothiol compounds useful in the methods of theinvention may be prepared by methods known in the art (see, e.g.,Cortese, 1943, Organic Synthesis pp. 91-93, Coll. Vol. II, Blatt, Ed.,John Wiley & Sons, Inc., New York, N.Y.; Akerfeldt, 1960, Acta Chem.Scand. 14:1980; Piper et al., 1966, Chem. Ind. (London):2010). Certainaminothiol compounds, as well as methods of synthesizing such compounds,are described in detail in U.S. Pat. Nos. 3,892,824, 5,424,472 and5,591,731, and WO 96/25045.

[0068] The aminothiol compounds useful in the methods of the inventionmay be in the form of free acids, free bases, or pharmaceuticallyacceptable addition salts thereof. Such salts can be prepared bytreating an aminothiol compound with an appropriate acid and/or base.Such acids include, by way of example and not limitation, inorganicacids such as hydrohalic acids (hydrochloric, hydrobromic, hydrofluoric,etc.), sulfuric acid, nitric acid, phosphoric acid, etc. and organicacids such as acetic acid, propanoic acid, 2-hydroxyacetic acid,2-hydroxypropanoic acid, 2-oxopropanoic acid, propandioic acid,butandioic acid, etc. Conversely, the salt can be converted into thefree base form by treatment with alkali.

[0069] The aminothiol compounds useful in the methods of the invention,as well as the pharmaceutically acceptable addition salts thereof, maybe in a hydrated, solvated or anhydrous form. Methods of preparing suchforms will be apparent to those of skill in the art of organicchemistry.

[0070] In another preferred embodiment, the compound is a salt ofWR-1065, preferably a succinate, a pamoate, a bis(trifluoroacetate) or adi-hydrochloric acid salt. The solubilities of these salts are shown inthe Examples section below.

6.1.2 Tissues to be Treated or Protected

[0071] It should be recognized that any and all tissue, skin or hair canbe treated or protected topically in accordance with the presentinvention. However, the preferred tissues to be treated or protectedaccording to the methods of the present invention are mucosal tissues.These tissues include, but are not limited to, alveolar, esophageal,gastric, gingival, laryngeal, lingual, nasal, olfactory, oral,pharyngeal, respiratory, tracheal and vaginal mucosa, as well as mucosaof the auditory tube, bronchi, ductus deferens, urethra, gallbladder,seminal vesicle, small intestine, tympanic cavity, ureter, urinarybladder and uterine tube. More preferred are mucosal tissues of the headand neck region, which include but are not limited to, esophageal,gingival, laryngeal, lingual, nasal, olfactory, oral, pharyngeal andtracheal mucosa, as well as mucosa of the tympanic cavity.

[0072] The invention is particularly well-suited to prevent or treatdamage to the mucosal tissues of the oral cavity to prevent or treatmucositis and related conditions and complications, including severedry-mouth known as xerostomia. Thus, oral mucosal tissues are mostpreferred.

[0073] The term “protect” as used herein means to avoid, reduce theincidence of, or reduce the severity of mucositis and related conditionsand complications and their symptoms.

[0074] The term “treat” as used herein means to lessen or reverse thesymptoms of mucositis and related conditions and complications.

6.1.3 Compositions and Formulations

[0075] The aminophosphorothioate or aminoalkyl thiol compounds describedherein, or pharmaceutically acceptable salts and/or hydrates thereof,may be administered singly, in combination with other compounds of theinvention, and/or in combination with other therapeutic agents,including cancer chemotherapeutic agents. The active compound(s) may beadministered alone or in the form of a pharmaceutical composition,wherein the active compound(s) is in admixture with one or morepharmaceutically acceptable carriers, excipients or diluents.

[0076] Suitable excipients, carriers and diluents are well known tothose skilled in the pharmaceutical arts, and include those which aresuitable for formulating topical preparations. When the tissues to betreated are mucosal tissues, including oral mucosa, suitable excipients,carriers and diluents must also be safe for application to oral andrelated mucosal tissue. These are well known to the skilled artisan.

[0077] Examples of excipients, carriers and diluents include, forexample, water, acetone, ethanol, ethylene glycol, propylene glycol,butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil,and mixtures thereof to form lotions, tinctures, creams, emulsions, gelsor ointments, which are non-toxic and pharmaceutically acceptable. Theskilled artisan can readily select the particular excipients, carriersand diluents appropriate to the type and location of the tissue to betreated.

[0078] Additionally, moisturizers or humectants can be added to thepresent composition if desired. Examples of such additional ingredientscan be found in Remington's Pharmaceutical Sciences, 16th and 18th eds.,Mack Publishing, Easton Pa. (1980 & 1990).

[0079] In addition to these and other vehicles which are known to thoseof ordinary skill in the art, it will be understood that thepharmaceutical compositions of the present invention may optionallyinclude other ingredients such as analgesics, anesthetics,antibacterial, antiyeast agents, antifungal agents, antiviral agents,antidermatitis agents, antipruritic agents, other anti-inflammatoryagents, antihyperkeratolytic agents, anti-dry skin agents, antipsoriaticagents, antisebborrheic agents, antihistamine agents, vitamins,corticosteroids, hormones, retinoids, topical cardiovascular agents,clotrimazole, ketoconazole, miconozole, griseofulvin, hydroxyzine,diphenhydramine, pratnoxine, lidocaine, procaine, mepivacaine,monobenzone, erythromycin, tetracycline, clindamycin, meclocyline,hydroquinone, minocycline, naproxen, ibuprofen, theophylline, cromolyn,albuterol, retinoic acid, 13-cis retinoic acid, hydrocortisone,hydrocortisone 21-acetate, hydrocortisone 17-valerate, hydrocortisone17-butyrate, betamethasone valerate, betamethasone diproprionate,triamcinolone acetonide, fluocinonide, clobetasol, proprionate, benzoylperoxide, crotamiton, propranolol, promethazine, vitamin A palmitate,vitamin E acetate and mixtures thereof.

[0080] Depending upon the specific tissue to be treated, additionalcomponents may be used prior to, in conjunction with, or subsequent totreatment with the active component(s). For example, penetrationenhancers may be used to assist in delivering the active component,e.g., amifostine, to the tissue. Suitable penetration enhancers includeacetone, various alcohols (e.g. ethanol, oleyl, tetrahydrofuryl, etc.),alkyl sulfoxides such as dimethyl sulfoxide, dimethyl acetamide,dimethyl formamide, polyethylene glycol, pyrrolidones such aspolyvinylpyrrolidone, Kollidon grades (Povidone, Polyvidone), urea, andvarious water-soluble or insoluble sugar esters such as Tween 80(polysorbate 80) and Span 60 (sorbitan monostearate).

[0081] The pH of the composition, or of the tissue to which thecompound(s) is applied, may also be adjusted to improve delivery of thecompound. Preferably, the pH is somewhat basic, as a basic pH isbelieved to enhance the stability of the active compounds. A pH of about8 to 9 is preferred. Similarly, the polarity of the solvent, its ionicstrength or tonicity may be adjusted to improve delivery. In addition,compounds such as stearates may be added to compositions comprising theactive compound(s) to advantageously alter the hydrophilicity orlipophilicity of the compound(s) and improve skin delivery. In thisregard, stearates can serve as a lipid vehicle for the formulation, asan emulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Also, different salts, hydrates or solvatesof the active compounds may be used to further adjust the properties ofthe resulting composition.

[0082] The compounds can be formulated in compositions such as creams,lotions, ointments, gels, solutions, suspensions or other forms known toone of skill in the art and described in, for example, Remington'sPharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa.(1980 & 1990), and Introduction to Pharmaceutical Dosage Forms, 4th ed.,Lea & Febiger, Philadelphia (1985). Most compositions of the presentinvention may be formulated as a solution, gel, lotion, cream orointment in a pharmaceutically acceptable form. Actual methods forpreparing pharmaceutical compositions are known or apparent to thoseskilled in the art and are described in detail in, for example,Remington's Pharmaceutical Sciences, 16th and 18th eds., MackPublishing, Easton Pa. (1980 & 1990).

[0083] In a preferred embodiment for use in treating mucosal tissueswithin the oral cavity, the active ingredient is formulated into asolution suitable for use as a mouthwash or as an oral gel.

[0084] Several preferred example formulations suitable for applicationto mucosal tissues are given in the Examples below.

6.1.4 Methods and Dosages

[0085] The compounds or pharmaceutical compositions containing them areapplied topically to the mucosal tissue. The term “topical” as usedherein includes any route of administration that enables the compoundsto line the mucosal tissue; i.e., it includes any route ofadministration other than parenteral or per os. Topical includes, forexample, spray application, smearing, soaking and gargling.

[0086] The specific form of the compounds and their manner ofadministration depends in part upon the particular tissue to be treated.The compounds or pharmaceutical compositions containing them can beapplied, for example, as a mouthwash to coat the oral mucosal tissue, asa spray or syringe to coat the mucosal tissues of the nose and/orthroat, or as a cream or paste, an enema, or other forms of topicaladministration known to one of skill in the art, as appropriate.

[0087] The amount of compound to be delivered, as well as the dosingschedule necessary to provide the desired radio/chemo protectiveeffects, will be dependent upon the bioavailability of the specificcompound selected (and/or an active metabolite thereof), the disorderbeing treated, the radiation and/or chemotherapeutic dosage schedule,and other factors that will be apparent to those of skill in the art.

[0088] When the compound is amifostine, preferably it will beadministered at a dosage of about 10 to 600 mg per dose, more preferablyat 100 to 300 mg per dose, and most preferably about 200 mg per dose. Itshould be understood that the preferred dosages are for localizedadministration, and if protection for a large amount of tissue isdesired, the dosage should be adjusted accordingly. The most preferreddosage of 200 mg per dose is appropriate, for example, for a typicalapplication such as a mouthwash to protect oral mucosal tissues.

[0089] For topical application, it is convenient to provide the activecompound in a pharmaceutical preparation at a concentration which willfacilitate easy application of the appropriate dosage, i.e., theconcentration should be chosen so that the volume of preparation to beapplied is not too great or too small. For typical treatment, aconvenient volume for topical application is about 1 to 30 mL perapplication. Thus, for example, when the desired dosage is 200 mg, it isconvenient to formulate the compound in a preparation at a concentrationof about 20 mg/mL, so that the desired dosage is administered bytopically applying about 10 mL of the preparation. However, inparticular situations it may be desirable to formulate the compound in asmaller or larger volume. One of skill in the art can readily determinea convenient concentration for a desired dosage.

[0090] While the dosage ranges given above are the preferred ranges foramifostine, these ranges should be adjusted according to the molecularweight of the active compound to deliver an equivalent number of moles.Thus, for example, a dosage of about 200 mg amifostine is equivalent toa dosage of about 130 mg WR-1065 if all other factors are equivalent.

[0091] The compounds may be administered prior to, during, or subsequentto radiation or chemotherapeutic treatment. Preferably, in order toprotect against tissue damage, the compounds will be administered to thetissue up to about 90 minutes prior to each radiation or chemotherapytreatment, and more preferably up to about 30 minutes prior to eachradiation or chemotherapy treatment.

[0092] The administration can be intermittent or continuous, and cancontinue or resume during or after treatment. Thus, for example, withspecific amounts and compounds given only by way of illustration, forthe treatment of oral mucosa about 10 mL of a mouthwash formulationcontaining about 20 mg/mL of amifostine may be administered to a patient5 to 30 minutes before irradiation or before administration of achemotherapeutic agent. The formulation can be rinsed and/or gargled inthe mouth for about 10 seconds to about 2 minutes, then spit out.Additional dosages administered in a similar manner can be given duringtreatment as, for example, between different chemotherapeutic agents ina regimen incorporating multiple chemotherapeutic agents per treatment.

[0093] Alternatively, the compound may be administered in a gel, lotion,ointment or other suitable form which is applied to the tissue up toabout 90 minutes before irradiation or chemotherapeutic treatment andremains on the tissue during the treatment.

[0094] The same dosage and concentrations can also be used when thecompound is administered after irradiation and/or chemotherapeutictreatment. It should be understood that each of the threeadministrations (before, during and after radio- and/or chemotherapytreatment) may be used alone, or in any combination of two or all threeadministrations, as needed.

6.2. Antibacterial Properties of Amifostine And Related Compounds

[0095] The present invention also encompasses methods of preventing andtreating infections, particularly those associated with mucositis, suchas secondary infections that occur as a result of radiation and/orchemotherapy. Bacterial infection of mucosal tissues is a common sideeffect of radiation and chemical damage associated with radio/chemocancer treatments. Damage to the mucosal basal epithelium, and resultantulceration, leaves the exposed underlying tissues extremely susceptibleto bacterial infection.

[0096] It is surprisingly discovered that amifostine and relatedcompounds are broad spectrum antibiotics as described in the UnitedStates Pharmacopeia, 23rd ed. (1995), “AntimicrobialPreservatives—Effectiveness” test protocol. Thus, pharmaceuticalpreparations containing amifostine and related compounds can beself-preserving. This self-preserving property is particularlyadvantageous for use in multi-dose formulations.

[0097] The antibacterial properties can also be used advantageously toprevent and treat infections, particularly those associated withmucositis, when the amifostine compounds are applied topically. Theantibacterial properties allow the topical use of the amifostinecompound after irradiation or chemotherapy to protect against bacterialinfection as well as symptoms of mucositis.

[0098] The types of secondary bacterial infections that can be readilytreated include gram negative, gram positive, yeast, mold andpseudomonas infections.

[0099] Certain embodiments of the invention are illustrated, and notlimited, by the following working examples.

7. EXAMPLES 7.1. Example 1 Effect of Topical Administration ofAmifostine on Radiation-induced Mucositis in Mice

[0100] The effect of topical amifostine on murine oral mucosa exposed toradiation was studied, to determine the radioprotective effects oftopically and systemically applied amifostine.

[0101] Materials and Methods

[0102] The experimental model used was the inferior lip mucosa of themice. The experimental murine model was developed by Parkins et al.,Radiother. Oncol., 1, 159-165 (1983), which has been shown to be areproducible tool to study mucosal reactions after irradiation in mice.C57BL/6 mice 8-10 weeks old were used and fed with semi-liquid food.Unanesthetized mice were maintained in supine position and irradiatedexclusively on the tip of the mouth. The mice were immobilized in jigscomparable to those described in Ang et al., Int. J. Radiat. Oncol.Biol. Phys., 8, 145-148 (1982). The irradiation was performed with an RT250 Phillips apparatus, delivering 1.98 Gy/min (200 kV, 20 mA, filter of0.2 mm Cu). During irradiation, a constant normobaric air renewal wasmaintained.

[0103] For the systemic applications, amifostine was dissolved in a 9%NaCl solution 5 minutes before systemic IP injection of 200 to 400mg/kg, which was administered 30 minutes before irradiation. A placebosolution of 9% NaCl was used for a control group. For the topicalapplications, 50 mg of amifostine was dissolved in 1 mL of a solutioncontaining 1% stearate, 5% H₂O, and ammonia to adjust the pH of thesolution to 8.8. The topical application was administered to the lipmucosa of the mice for a period of 30 minutes before each radiationsession, and was maintained throughout the duration of irradiation. Asingle dose of 16.5 Gy, and a fractionated schedule delivering 24 Gy in4 fractions over 2 days (with 8 hour intervals between fractions) wereadministered.

[0104] The effect of irradiation on lip mucosa was evaluated using thescoring system described by Parker, and shown in Table 1. TABLE 1Parkins scoring system Score Mucosal Observation/Erythema 0.5 doubtfulif abnormally pink 1 slight but definite reddening 2 severe reddening 3focal desquamation 4 exudation or crusting covering about ½ lip area 5exudation or crusting covering more than ½ lip area Separate score foredema (swelling) of the lips, to be added to the erythema scores givinga maximum score of 7: 0.5 50-50 doubtful if any swelling 1 slight butdefinite swelling 2 severe swelling

[0105] Four groups of eight mice were scored and weighed each day. Anymice having lost 30% or more of the initial weight was sacrificed. Theeffect of mucositis and weight loss were compared at the maximum of theacute reactions (day 11) among the different groups receiving or notreceiving amifostine treatments, using the Mann and Whitney test.

[0106] Results

[0107] In a first set of experiments, the mice were irradiated with asingle dose of 16.5 Gy. The distribution of weight loss and of the scoreof mucosal reactions after IP and topical administration is shown inFIG. 1. The maximum mucosal reactions were observed on day 11, followingthe start of irradiation. The mucosal reaction grades of the mice areshown in FIG. 2. The grades of mucosal reactions were significantlylower in the groups receiving amifostine compared to the control group.The Figure shows that the mucosal grades for IP administered amifostineare lower, but the difference was not found to be statisticallysignificant.

[0108] Similar results were found in a second experiment, in which afractionated schedule of 24 Gy in 4 fractions over 2 days was deliveredusing two different levels of amifostine IP: 200 mg/kg and 400 mg/kg.400 mg/kg given before each fraction of radiation was the maximumtolerated dose. In this experiment, the topical amifostine wasadministered as described above without modification.

[0109] The maximum grade of mucosal reactions occurred on day 11 afterirradiation, and was found to be 3.5±0.2 and 2.5±0.4 for the 200 and 400mg/kg IP dosages, respectively. The error bars represent the standarderror. For topical amifostine, the maximum grade of mucositis was foundto be 3.9±0.2, and was not statistically significantly different fromthe IP groups. The control group receiving no amifostine showed amucosal reaction grade of 5.6±0.4, and was statistically different(p=0.001) from the amifostine groups.

7.2. Example 2 Antibacterial Properties of Amifostine

[0110] Amifostine was studied for its ability to inhibit microbialgrowth. Clear 10 cc vials, stoppers and seals were sterilized for use inthe study. Two separate procedures were performed: a refrigeratedstorage study (2° to 8° C.) and a room temperature storage study (20° to25° C.). For each test portion, positive controls were prepared for eachorganism by placing 9.5 mL sterile 0.9% sodium chloride into separatesterile vials and inoculating each with 10³ to 10⁴ of one of the testorganisms. In addition, amifostine test specimens were prepared for eachtest portion by reconstituting six Ethyol® vials with 0.9% sodiumchloride, the contents of each of these vials were then transferred toclear sterile vials to allow for easier visual examination. One vial wasdesignated as a negative control, and each of the remaining five vialswas inoculated with 10³ to 10⁴ of one of the test ;organisms. All vialswere protected from light throughout the test to simulate the amberEthyol® vial.

[0111] Test Performance

[0112] Refrigerated Storage (2°-8° C.)

[0113] Positive controls, product test specimens, and a negative controlwere prepared. At this time, population counts were performed on eachpositive control. Population counts were also performed on positivecontrols and product test specimens at 24, 48 and 72 hours. Thesesamples were diluted with sterile water and plated in duplicate with atarget of 30 to 300 colonies per plate. Candida albicans, Escherichiacoli, Pseudomonas aeruginosa, and Staphylococcus aureus were overpouredwith TSA agar and incubated at 30-35° C. for 48 hours. Aspergillus nigerwas overpoured with Sabouraud agar and incubated at 20-25° C. for 48hours. The plates were counted and the population counts are summarizedin Table 2. TABLE 2 Refrigerated Storage (2°-8° C.) Time CFU/mL inCFU/mL in (hr) Amifostine % Charge Saline % Charge S. aureus 0 9.50 ×10^(1*) 9.50 × 10¹ 24 4.75 × 10¹ −50.0% 4.75 × 10¹ −50% 48 4.75 × 10¹−50.0% 4.75 × 10¹ −50% 72 4.75 × 10¹ −50.0% 4.75 × 10¹ −50% P.aeruginosa0 3.80 × 10^(3*) 3.80 × 10³ 24 3.04 × 10³ −20.0 3.47 × 10³ −0.08 48 1.76× 10³ −53.7 1.48 × 10³ −61.0 72 1.85 × 10³ −51.3 2.23 × 10³ −41.3 E.coli0 9.50 × 10^(2*) 9.50 × 10² 24 2.85 × 10² −70.0 1.90 × 10² −80.0 48 0−100 4.75 × 10¹ −95.0 72 0 −100 0 −100 C.albicans 0 1.08 × 10^(4*) 1.08× 10⁴ 24 3.99 × 10³ −63.0 7.70 × 10³ −28.7 48 2.09 × 10³ −80.6 5.52 ×10³ −48.9 72 7.13 × 10³ −34.0 5.70 × 10³ −47.2 A.niger 0 3.00 × 10^(5*)3.00 × 10⁵ 24 1.78 × 10⁵ −40.7 1.57 × 10⁵ −47.7 48 1.67 × 10⁵ −44.3 1.38× 10⁵ −54.0 72 1.67 × 10⁵ −44.3 1.53 × 10⁵ −49.0

[0114] At 48 hours, the concentrations of viable microorganisms werereduced by ≧44.3% of the initial concentration. Incubation for anadditional 24 hours demonstrated continued loss of viability.

[0115] Room Temperature Storage (20°-25° C.)

[0116] Positive controls, product test specimens, and a negative controlwere prepared. At this time, population counts were performed on eachpositive control. Population counts were also performed on positivecontrols and product test specimens at 24 and 48 hours. These sampleswere diluted with sterile water and plated in duplicate with a target of30 to 300 colonies per plate. Candida albicans, Escherichia coli,Pseudomonas aeruginosa, and Staphylococcus aureus were overpoured withTSA agar and incubated at 30-35° C. for 48 hours. Aspergillus niger wasoverpoured with Sabouraud agar and incubated at 20-25° C. for 48 hours.The plates were counted and the population counts are summarized inTable 3. TABLE 3 Room Temperature Storage (20°-25° C.) Time CFU/mL inCFU/mL in (hr) Amifostine % Charge Saline % Charge S.aureus 0 1.00 ×10^(5*) 1.00 × 10⁵ 24 9.60 × 10⁴ −4.0% 1.01 × 10⁵ 0% 48 3.32 × 10²−99.7% 0 −100% P.aeruginosa 0 2.61 × 10^(3*) 2.61 × 10² 24 5.22 × 10²−80.0 2.09× 10³ −21.0 48 0 −100 1.33 × 10³ −49.0 E.coli 0 2.79 × 4* 2.79× 10⁴ 24 2.28 × 10³ −91.8 2.90 ×10³ −89.6 48 8.55 × 10² −96.9 9.50 × 10¹−99.6 C. albicans 0 1.66 × 10^(4*) 1.66 × 10⁴ 24 1.43 × 10⁴ −13.8 1.66 ×10⁴ 0 48 1.00 × 10⁴ −39.8 1.44 × 10⁴ −13.2 A. niger 0 9.10 × 10^(6*)9.10 × 10⁴ 24 1.56 × 10⁷ +58.3 1.73 × 10⁷ +52.6 48 3.52 × 10⁶ −61.3 5.46× 10⁶ −40.0

[0117] At 48 hours, a population count was performed on the negativecontrol and all test samples were subcultured for microbialidentification. Results revealed that the negative control was absent ofmicrobial growth and all test specimens contained the stated testorganisms.

[0118] At 24 hours, the concentration of viable bacteria were reduced infour of the five compendial organisms. The concentration of A. nigerincreased at 24 hours, but decreased significantly at 48 hours.

7.3. Example 3 Antibacterial Properties of WR-1065

[0119] WR-1065 was evaluated according to a protocol based on the“Antimicrobial Preservatives-Effectiveness” Test, outlined in the UnitedStates Pharmaeopeia, 23rd ed. (1995).

[0120] Procedure

[0121] Preparation of Mold Spore Inoculum Suspension

[0122] A sterile swab is used to spread 1.0 mL of a spore suspensionover the surface of solidified SDA in a 150×15 mm Petri dish so as toresult in a confluent lawn of mold growth. The culture is then incubatedat 20-25° C. for 7 days to allow for mature spore development. Followingincubation, the spores are harvested in approximately 25 mL of sterile0.9% Saline with Polysorbate® 80 by spreading the fluid across thesurface of the culture and gently scraping with a sterile spoonula todislodge the spores. The resulting suspension is transferred into asterile tissue grinder and the piston is reciprocated several times tobreak up spore chains. A spore count is obtained with the aid of ahemocytometer and the spore concentration is adjusted, if necessary, toapproximately 1.0×10⁸ spores/mL.

[0123] Preparation of the Bacterial and Yeast Inoculum Suspensions

[0124] Bacterial challenge organisms are inoculated onto TSA slants fromstock cultures and incubated for 18-24 hours at 30-35° C. The yeastchallenge organism is inoculated onto a SDA slant from stock culture,and incubated for 48 hours at 20-25° C. Following incubation, organismsare harvested from the agar surfaces in 0.9% Saline. Cell population ineach suspension is estimated by determining percent transmittance on aspectrophotometer and consulting established growth curves for eachorganism. Each suspension is adjusted to an approximate concentration of1.0×10⁸ Colony Forming Units (CFu) per mL. The number of viablemicroorganisms in each inoculum suspension is determined by plate countprocedure, and the initial concentration of microorganisms per gram (ormL) of WR-1065 is calculated. The numbers obtained from this procedureprovide the theoretical concentration of challenge organisms present ineach test article at the start of the study, and are used as thebaseline for calculating the reduction of the number of organisms overtime.

[0125] Neutralization

[0126] The neutralizer medium used as a diluent is AOAC NeutralizerBlanks. MCT agar is used as the plating medium for all bacteriarecovery. SDA/L is used as the plating medium for fungal recovery.

[0127] A neutralization study is conducted to evaluate the preservativeneutralizing ability of the media used to conduct the test and to assurethat viable organisms can be detected under the conditions of the test.Separate 1:10 (and 1:100, if necessary) dilutions of WR-1065 areprepared in diluent for each test organism. A blank control of the samevolume is also prepared for each organism. Each dilution and controltube is inoculated with 0.1 mL of organism suspension to result in alevel of approximately 10-100 CFU/mL of suspension. Plate counts areobtained on each suspension. After incubation, colony forming units areenumerated and percent recovery between control plates and test platesis evaluated. Neutralization is effective if 50% or greater recovery isobserved on test plates as compared to the inoculum control plates.

[0128] Plating and Plate Incubation

[0129] Plates prepared in this study are incubated according to thefollowing standard:

[0130] MCT plates for bacteria recovery: 2-3 days at 30-35° C.

[0131] SDA/L plates for fungal recovery: 5-7 days at 20-25° C.

[0132] All plating for this study is conducted in duplicate.

[0133] Initial Count Procedure

[0134] Prior to inoculation of the test article, a 1:10 dilution isprepared in diluent and 1.0 mL is plated. Additional serial dilutionsare prepared and plated as necessary. Two separate sets are prepared asabove. One set is prepared for bacteria counts, and one set is preparedfor yeast/mold counts.

[0135] Inoculation of Test Article

[0136] Five separate 20 g (or 20 mL, as appropriate) portions of thetest article are placed into sterile 50 mL polypropylene conical tubesand inoculated with 0.1 mL of individual inoculum suspension to resultin a final concentration of between 1.0×10⁵ and 1.0×10⁶ CFU/g (or mL) oftest article. All inoculated specimens are vigorously mixed to assureeven dispersal of the inoculum. Inoculated test articles are held at20-25 ° C. for the duration of the study.

[0137] Post Inoculation Procedure

[0138] Enumeration of viable bacterial and fungal populations in theinoculated test article is conducted at 7, 14, 21 and 28 days postinoculation. At each time interval, a 1.0 g (or mL) specimen is removedfrom each inoculated specimen and plate counts are obtained by preparingserial dilutions in 9 mL tubes of diluent and plating aliquots from eachdilution. Following incubation, plates are observed and the CFU's areenumerated. The number obtained is multiplied by the dilution factor togive the number of viable organisms per g (or mL) of test article ateach time interval. At each time interval, the inoculated product isexamined and any changes observed in the appearance of the test articleduring the challenge period are recorded.

[0139] WR-1065 was tested according to the procedure described above.The results are shown in Table 4. TABLE 4 Test Organism Recovery (CFU/g)Test Organism Inoculum Counts* 7 Day 14 Day 21 Day 28 Day E. coil ATCC8739 4.9 × 10⁵ <10 <10 <10 <10 P. aeruginosa ATCC 9027 5.5 × 10⁵ <10 <10<10 <10 S. aureus ATCC 6538 4.1 × 10⁵ 8.8 × 10⁴ <10 <10 <10 C. albicansATCC 10231 4.5 × 10⁵ 3.0 × 10⁴ <10 <10 <10 A. niger ATCC 16404 3.5 × 10⁵1.7 × 10⁵ 1.3 × 10⁵ 1.2 × 10⁶ 7.5 × 10⁴

[0140] The initial counts of bacteria, yeast and mold were <10 CFU/g.

[0141] Interpretation

[0142] The efficacy of WR-1065 was evaluated according to the followingcriteria stated in the United States Pharmacopeia, USP 23, 1995:

[0143] The preservative is effective in the product examined if: (a) theconcentrations of viable bacteria are reduced to not more than 0.1% ofthe initial concentration by the 14th day; (b) the concentrations ofviable yeast and mold remain at or below the initial concentrationsduring the first 14 days; and (c) the concentration of each testorganism remains at or below these designated levels during theremainder of the 28 day test period.

[0144] According to these criteria, WR-1065 is an effective preservativefor each of the tested microorganisms.

7.4. Example 4 Solubility of WR-1065 Salts

[0145] As mentioned above, salts of amifostine and related compounds areincluded within the scope of the invention. Certain salts may haveadvantages in topical administration. Several salts of WR-1065 wereprepared, and their solubility in various solvents was measured. Thedata are shown in Table 5. TABLE 5 Solubility of WR-1065 Salts Est.Solubitity Est. Sotubitity at Saturation HPLC Assay Salt M.W. (g/mol)Solvent (mg/mL) (mg/mL) Succinate 252 Water 854 715 Succinate (as 455381 free base) Succinate Ethanol insoluble 1.95 Succinate (as 1.04 freebase) Pamoate 522.6 70% isopropyl 21 18.5 alcohol Pamoate (as free 5.344.8 base) Pamoate Ethanol insoluble 0.2 Pamoate (as free 0.05 base) BisTFA* 362.3 Water 820 954 Bis TEA* (as free 354 353 base) Bis TFA*Ethanol 313 327 Bis TFA (as free 116 121 base) di-HCl Water 849 di-HCL(as free 550 base)

7.5. Example 5 Formulations

[0146] Several pharmaceutical ointments were prepared for use withamifostine for topical application to oral mucosal tissue. Each of theseformulations has a pH of 8.8, adjusted by addition of ammonia. Theointment bases are summarized in Table 6. TABLE 6 Ointment BaseCompositions Base # Component Amount 1 Tween 80 5 mL Glycerol starch*100 g Water (sterile, demineralized) 5 mL Ammonia to pH 8.8 2 Tween 80 5mL Glycerol starch 100 g Stearates 1 g Ammonia to pH 8.8 3 Tween 80 5 mLGlycerol starch* 100 g Stearates 1.5 g Ammonia to pH 8.8 4 Lactose 10 gWater (sterile, demineralized) 10 mL Petrolatum 60 g Ammonia to pH 8.8

[0147] Tween 80 is a water-soluble ester (polyoxyethylene oxide sorbitanmonooleate, or polysorbate 80). Glycerol starch is a gelatinous mixtureof glycerol and starch, suitable as a base for a gel or ointment. Othersimilar components may be used as well.

[0148] Compositions according to the present invention were prepared byfirst mixing amifostine in a minimal quantity (40 μL for 50 mg ofamifostine) of a 9% sodium chloride solution, then combining with theointment base. Formulations were prepared at amifostine concentrationsof 20, 100 and 200 mg/mL of ointment base.

[0149] The invention described and claimed herein is not to be limitedin scope by the specific embodiments herein disclosed, since theseembodiments are intended as illustrations of several aspects of theinvention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims.

[0150] All references cited in the present application are incorporatedby reference in their entirety.

What is claimed is:
 1. A method of protecting tissue of a mammal fromdamage caused by radiation and/or chemotherapeutic treatment, whichcomprises topically administering to the tissue of the mammal atherapeutically effective amount of at least one compound having theformula R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂ wherein R₁ is hydrogen, C₁-C₇ aryl,C₁-C₇ acyl, or C₁-C₇ alkyl; R₂ is hydrogen, PO₃H₂ or R₃, wherein R₃ isR₁NH(CH₂)_(n)NH(CH₂)_(m)S—; n is an integer from 1 to 10; and m is aninteger from 1 to 10, or a pharmaceutically acceptable salt, solvate orhydrate thereof.
 2. A method of protecting mucosal tissue in the headand/or neck region of a mammal from mucositis and the symptoms thereof,which comprises topically administering to the mucosal tissue of themammal a therapeutically effective amount of at least one compoundhaving the formula R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂ wherein R₁ is hydrogen,C₁-C₇ aryl, C₁-C₇ acyl, or C₁-C₇ alkyl; R₂ is hydrogen, PO₃H₂ or R₃,wherein R₃ is R₁NH(CH₂)_(n)NH(CH₂)_(m)S—; n is an integer from 1 to 10;and m is an integer from 1 to 10, or a pharmaceutically acceptable salt,solvate or hydrate thereof.
 3. The method of claim 2, wherein R₁ ishydrogen or methyl, R₂ is hydrogen or PO₃H₂, m is 2 or 3,and n is
 3. 4.The method of claim 2, wherein the compound is amifostine, WR-1065,WR-151326, WR-151327, or WR-33278, or a pharmaceutically acceptablesalt, solvate or hydrate thereof.
 5. The method of claim 4, wherein thecompound is amifostine, or a pharmaceutically acceptable salt, solvateor hydrate thereof.
 6. The method of claim 4, wherein the compound isWR-1065, or a pharmaceutically acceptable salt, solvate or hydratethereof.
 7. The method of claim 2, wherein the compound is administeredin the form of a pharmaceutical composition comprising the compound anda pharmaceutically acceptable excipient, carrier or diluent.
 8. Themethod of claim 7, wherein the composition further comprises apenetration enhancing agent or a pH adjusting agent.
 9. The method ofclaim 2, wherein the mucositis is induced at least in part by radiation.10. The method of claim 9, wherein the compound is administered to themucosal tissue of the mammal prior to radiation treatment, duringradiation treatment, or both prior to and during radiation treatment.11. The method of claim 10, wherein the compound is administered to themucosal tissue no more than about 90 minutes prior to each radiationtreatment.
 12. The method of claim 10, wherein the compound isadministered in a dosage of 10 to 600 mg.
 13. The method of claim 12,wherein the compound is administered in a dosage of 100 to 300 mg. 14.The method of claim 2, wherein the mucosal tissue is esophageal mucosa,gingival mucosa, laryngeal mucosa, lingual mucosa, nasal mucosa,olfactory mucosa, oral mucosa, pharyngeal mucosa, tracheal mucosa ormucosa of the tympanic cavity.
 15. The method of claim 14, wherein themucosal tissue is oral mucosa.
 16. The method of claim 2, wherein themucositis is induced at least in part by treatment with chemotherapeuticagents.
 17. The method of claim 16, wherein the compound is administeredto the mucosal tissue of the mammal prior to treatment withchemotherapeutic agents, during treatment with chemotherapeutic agents,or both prior to and during treatment with chemotherapeutic agents. 18.The method of claim 17, wherein the compound is administered to themucosal tissue no more than 90 minutes prior to each chemotherapytreatment.
 19. The method of claim 18, wherein the compound isadministered in a dosage of 10 to 600 mg.
 20. The method of claim 19,wherein the compound is administered in a dosage of 100 to 300 mg.
 21. Amethod of protecting oral mucosal tissue in a mammal from mucositis orthe symptoms of mucositis induced at least in part by radiation, whichmethod comprises topically administering to the oral mucosa of themammal a therapeutically effective amount of a compound selected fromthe group consisting of amifostine, WR-1065, pharmaceutically acceptablesalts, solvates and hydrates of amifostine and WR-1065, and mixturesthereof, prior to, during, or both prior to and during exposure toradiation.
 22. The method of claim 21, wherein the compound isadministered to the oral mucosa of the mammal in the form of apharmaceutical composition comprising the compound and apharmaceutically acceptable excipient, carrier or diluent.
 23. Themethod of claim 22, wherein the composition further comprises apenetration enhancing agent or a pH adjusting agent.
 24. The method ofclaim 21, wherein the compound is administered to the mucosal tissue nomore than 90 minutes prior to each radiation treatment.
 25. The methodof claim 21, wherein the compound is administered in a dosage of 10-600mg.
 26. The method of claim 25, wherein the compound is administered ina dosage of 100-300 mg.
 27. A method of treating damaged or infectedmucosal tissue of a mammal, which comprises topically administering tothe mucosal tissue of the mammal a therapeutically effective amount ofat least one compound having the formula R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂wherein R₁ is hydrogen, C₁-C₇ aryl, C₁-C₇ acyl, or C₁-C₇ alkyl; R₂ ishydrogen, PO₃H₂ or R₃, wherein R₃ is R₁NH(CH₂)_(n)NH(CH₂)_(m)S—; n is aninteger from 1 to 10; and m is an integer from 1 to 10, or apharmaceutically acceptable salt, solvate or hydrate thereof.
 28. Themethod of claim 27, wherein the compound is administered to the damagedmucosal tissue of the mammal in the form of a pharmaceutical compositioncomprising the compound and a pharmaceutically acceptable excipient,carrier or diluent.
 29. The method of claim 28, wherein the compositionfurther comprises a penetration enhancing agent or a pH adjusting agent.30. The method of claim 27, wherein the compound is administered in adosage of 10-600 mg.
 31. The method of claim 30, wherein the compound isadministered in a dosage of 100-300 mg.
 32. A method of treating orpreventing bacterial infection in a mammal which comprises topicallyadministering to the mammal a therapeutically effective amount of atleast one compound having the formula R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂wherein R₁ is hydrogen, C₁-C₇ aryl, C₁-C₇ acyl, or C₁-C₇ alkyl; R₂ ishydrogen, PO₃H₂ or R₃, wherein R₃ is R₁NH(CH₂)_(n)NH(CH₂)_(m)S—; n is aninteger from 1 to 10; and m is an integer from 1 to 10, or apharmaceutically acceptable salt, solvate or hydrate thereof.
 33. Themethod of claim 32 wherein said mammal is a human.
 34. The method ofclaim 33 wherein said human is undergoing radiation therapy orchemotherapy.
 35. The method of claim 32 wherein said human hasundergone radiation therapy or chemotherapy.
 36. The method of claim 32,wherein R₁ is hydrogen or methyl, R₂ is hydrogen or PO₃H₂, m is 2 or 3,and n is
 3. 37. The method of claim 32, wherein the compound isamifostine, WR-1065, WR-151326, WR-151327, or WR-33278, or apharmaceutically acceptable salt, solvate or hydrate thereof.
 38. Themethod of claim 37, wherein the compound is amifostine, or apharmaceutically acceptable salt, solvate or hydrate thereof.
 39. Themethod of claim 32, wherein the compound is WR-1065, or apharmaceutically acceptable salt, solvate or hydrate thereof.
 40. Themethod of claim 32, wherein the compound is administered in the form ofa pharmaceutical composition comprising the compound and apharmaceutically acceptable excipient, carrier or diluent.
 41. Aself-preserving multi-dose pharmaceutical composition comprisingamifostine, WR-1065, or a pharmaceutically acceptable salt, solvate orhydrate thereof.